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1,25-‐Dihydroxyvitamin D3 and Re7noic Acid in vitro Modula7on of PPARγ and Insulin Resistance in

Diabetes Mellitus II

Brian Covello

Mo7va7on •  25.8 million people affected by DM •  By 2050, 1 in 3 US adults will be affected •  Normal human physiology gone wrong – Glucose à Insulin

•  Beta cells, islet of Langerhans – Type I and Type II

•  Linked to Obesity •  TZDs as an7-‐diabe7c drugs

PPARγ

•  RXR green •  PPARγ purple

•  57.6kDa •  Adipogenesis, Insulin

sensi7vty

1,25-‐Dihydroxyvitamin D3 and Re7noic Acid in vitro Modula7on of PPARγ and Insulin Resistance in

Diabetes Mellitus II

Brian Covello

•  Limited supply of RXR •  VDR and RAR heterodimerize with RXR

•  Fierce compe77on! •  Ligands – VDR = D3 – RAR = RA

The Connec7on

What Do We Know?

•  Vitamin D down regulates expression of PPARγ (Yoshifumi, 1998) – Vitamin D correlated to Obesity

•  Re7noic Acid also inhibits PPARγ (Yoshifumi, 1998)

•  RA and D3 effects tested separately •  Muta7ons in PPARγ cause insulin resistance (Barroso, 1999)

The Research •  Diabetes Mellitus Type II •  In vitro analysis, in vivo predic7ons •  A bridge to transla7onal research •  Mimic human physiology •  D3 and RA tested together – first 7me! •  Nanomolar human serum concentra7on

•  Hypothesis: Synergy of down regula7on of PPARγ when D3 and RA are combined

•  Implica7ons: Diet & Drug interac7on

Bidirec7onal Approach

•  Immunofluorescence Microscopy – Delocaliza7on of PPARγ –  IF never conducted in this manner – Unable to indicate up/down regula7on

•  SDS-‐PAGE – Separa7on of proteins based on size – Protein changes? – Up/down regula7on à Western Blot

Immunofluorescence Microscopy

•  Fixed à Permeabilized à Blocked •  Primary AB = rabbit an7-‐PPAR

–  BD transduc7on labs –  1:25 concentra7on

•  Secondary AB = goat an7-‐rabbit SFX kit –  Alexa Fluora, 1:25

•  + control 1% EtOH à delocaliza7on (Powers, 2005)

•  -‐ Control Incomplete

Tx 1: Incomplete Tx 2: 1% EtOH Tx 3: 1 μM D3 Tx 4: 1 μM RA Tx 5: 1 nm D3 Tx 6: 1 nm RA Tx 7: 1 μM D3/RA Tx 8: 1 nM D3/RA

I Swear… I didn’t kill them

•  Proof: •  Elongated •  Nuclei present and dis7nct •  Amount of cells per frame the same

•  40X •  Canon •  B. Covello

SDS-‐PAGE

•  Gives false nega7ve charge to proteins •  Separated based on size •  Proteins migrate toward + end •  50mA •  Make sure to put same protein amount in each well…

Standard Curve

•  50 micrograms

•  Disappearance of protein in combined •  Novel protein changes in nanomolar

treatment that aren’t present in micromolar •  Human serum levels

Where Do We Go From Here?

•  Western Blot – What are those proteins? –  PPARγ changes?

•  3T3-‐L1 –  Pre-‐adipocytes –  Closer to human physiology –  Berer rela7on to DMII – Differen7a7on process

•  Insulin, Dexamethosone, 3-‐isobutyl-‐1-‐methylxanthine (IBMX)

Acknowledgements

•  FSC for funding •  Dr. Bradshaw •  Classmates

Sources Barroso, I. B. (1999). Dominant nega7ve muta7ons in human ppar gamma associated with sever insulin resistance, diabetes

mellitus and hypertension. Le+ers to Nature, 402(23), 880-‐889. Liang, G. L. (2006). Peroxisome proliferator ac7vated receptor gamma as a drug target in the pathogenesis of insulin resistance.

Pharmacology & Therapeu=cs, 111(4), 145-‐173. Ishida, Y. (1988). Possible involvement of 1,25-‐dihydroxyvitamine d3 in prolifera7on and differen7a7on of 3t3-‐l1 cells.

Biochemical and Biophysical Research Communica=ons, 151(3), 1122-‐1127. Yoshifumi. (1998). Counterac7on of re7noic acid and 1,25-‐dihydroxyvitamin d3 on up-‐regula7on of adipocyte differen7a7on with

ppar ligand, an an7diabe7c thiazolidinedione, in 3t3-‐l1 cells. Pharmacology Le+ers, 62(14), 205-‐211. Powers, A. C. (2005). Chapter 323. Diabetes mellitus. In D. L. Kasper, A. S. Fauci, D. L. Longo, E. Braunwald, S. L. Hauser, & J. L.

Jameson, Harrison’s principles of internal medicine (16th ed.). The McGraw-‐Hill Companies, Inc. Wajchenberg, B. L. (2000). Subcutaneous and visceral adipose 7ssue: their rela7on to the metabolic syndrome. Endocr Rev 21,

697–738. Bogazzi, F. (2007). Abnormal expression of ppar gamma isoforms in the subcutaneous adipose 7ssue of pa7ents with cushing's

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